EP1987150B1 - Selection of host cells expressing protein at high levels - Google Patents

Selection of host cells expressing protein at high levels Download PDF

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EP1987150B1
EP1987150B1 EP07712283A EP07712283A EP1987150B1 EP 1987150 B1 EP1987150 B1 EP 1987150B1 EP 07712283 A EP07712283 A EP 07712283A EP 07712283 A EP07712283 A EP 07712283A EP 1987150 B1 EP1987150 B1 EP 1987150B1
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polypeptide
selectable marker
sequence
interest
expression
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EP1987150A2 (en
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Arie Pieter Otte
Henricus Johannes Maria Van Blokland
Theodorus Hendrikus Jacobus Kwaks
Richard George Antonius Bernardus Sewalt
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Chromagenics BV
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Chromagenics BV
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Priority claimed from US11/359,953 external-priority patent/US20060141577A1/en
Priority claimed from US11/416,490 external-priority patent/US20060195935A1/en
Application filed by Chromagenics BV filed Critical Chromagenics BV
Priority to EP07712283A priority Critical patent/EP1987150B1/en
Priority to PL07712283T priority patent/PL1987150T3/pl
Priority to SI200730698T priority patent/SI1987150T1/sl
Publication of EP1987150A2 publication Critical patent/EP1987150A2/en
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/40—Vector systems having a special element relevant for transcription being an insulator
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    • C12N2840/00—Vectors comprising a special translation-regulating system
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    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
    • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES

Definitions

  • the invention relates to the field of molecular biology and biotechnology. More specifically the present invention relates to means and methods for improving the selection of host cells that express proteins at high levels.
  • Proteins can be produced in various host cells for a wide range of applications in biology and biotechnology, for instance as biopharmaceuticals.
  • Eukaryotic and particularly mammalian host cells are preferred for this purpose for expression of many proteins, for instance when such proteins have certain posttranslational modifications such as glycosylation.
  • Methods for such production are well established, and generally entail the expression in a host cell of a nucleic acid (also referred to as 'transgene') encoding the protein of interest.
  • a nucleic acid also referred to as 'transgene'
  • the transgene together with a selectable marker gene is introduced into a precursor cell, cells are selected for the expression of the selectable marker gene, and one or more clones that express the protein of interest at high levels are identified, and used for the expression of the protein of interest.
  • transgenes One problem associated with the expression of transgenes is that it is unpredictable, stemming from the high likelihood that the transgene will become inactive due to gene silencing (McBurney et al., 2002), and therefore many host cell clones have to be tested for high expression of the transgene.
  • bicistronic expression vectors have been described for the rapid and efficient creation of stable mammalian cell lines that express recombinant protein.
  • These vectors contain an internal ribosome entry site (IRES) between the upstream coding sequence for the protein of interest and the downstream coding sequence of the selection marker (Rees et al, 1996).
  • IRS internal ribosome entry site
  • Such vectors are commercially available, for instance the pIRES1 vectors from Clontech ( CLONTECHniques, October 1996 ).
  • selection of sufficient expression of the downstream marker protein then automatically selects for high transcription levels of the multicistronic mRNA, and hence a strongly increased probability of high expression of the protein of interest is envisaged using such vectors.
  • the IRES used is an IRES which gives a relatively low level of translation of the selection marker gene, to further improve the chances of selecting for host cells with a high expression level of the protein of interest by selecting for expression of the selection marker protein (see e.g. WO 03/106684 and WO 2006/005718 ).
  • the present invention aims at providing improved means and methods for selection of host cells expressing high levels of proteins of interest.
  • WO 2006/048459 was filed before but published after the priority date of the instant application, WO 2006/048459 discloses a concept for selecting host cells expressing high levels of polypeptides of interest, the concept referred to therein as 'reciprocal interdependent translation'.
  • a multicistronic transcription unit is used wherein a sequence encoding a selectable marker polypeptide is upstream of a sequence encoding a polypeptide of interest, and wherein the translation of the selectable marker polypeptide is impaired by mutations therein, whereas translation of the polypeptide of interest is very high (see e.g. FIG. 13 therein for a schematic view).
  • the present invention provides alternative means and methods for selecting host cells expressing high levels of polypeptide.
  • the invention provides a. DNA molecule comprising a multicistronic transcription unit coding for i) a polypeptide of interest, and for ii) a selectable marker polypeptide functional in a eukaryotic host cell, wherein the polypeptide of interest has a translation initiation sequence separate from that of the selectable marker polypeptide, and wherein the coding sequence for the polypeptide of interest is upstream from the coding sequence for the selectable marker polypeptide in said multicistronic transcription unit, and wherein an internal ribosome entry site (IRES) is present downstream from the coding sequence for the polypeptide of interest and upstream from the coding sequence for the selectable marker polypeptide, and wherein the nucleic acid sequence coding for the selectable marker polypeptide in the coding strand comprises a translation start sequence chosen from the group consisting of: a) a GTG startcodon; b) a TTG startcodon; c) a CTG startcodon; d) a ATT
  • the translation start sequence in the coding strand for the selectable marker polypeptide comprises a startcodon different from an ATG startcodon, such as one of GTG, TTG, CTG, ATT, or ACG sequence, the first two thereof being the most preferred.
  • ATG startcodon such as one of GTG, TTG, CTG, ATT, or ACG sequence, the first two thereof being the most preferred.
  • non-ATG startcodons preferably are flanked by sequences providing for relatively good recognition of the non-ATG sequences as startcodons, such that at least some ribosomes start translation from these startcodons, i.e. the translation start sequence preferably comprises the sequence ACC[non-ATG startcodon]G or GCC[non-ATG startcodon]G.
  • the selectable marker protein provides resistance against lethal and/or growth-inhibitory effects of a selection agent, such as an antibiotic.
  • the invention further provides expression cassettes comprising a DNA molecule according to the invention, which expression cassettes further comprise a promoter upstream of the multicistronic expression unit and being functional in a eukaryotic host cell for initiation transcription of the multicistronic expression unit, and said expression cassettes further comprising a transcription termination sequence downstream of the multicistronic expression unit.
  • such expression cassettes further comprise at least one chromatin control element chosen from the group consisting of a matrix or scaffold attachment region (MAR/SAR), an insulator sequence, a ubiquitous chromatin opener element (UCOE), and an anti-repressor sequence.
  • chromatin control element chosen from the group consisting of a matrix or scaffold attachment region (MAR/SAR), an insulator sequence, a ubiquitous chromatin opener element (UCOE), and an anti-repressor sequence.
  • Anti-repressor sequences are preferred in this aspect, and in certain embodiments said anti-repressor sequences are chosen from the group consisting of: a) any one SEQ. ID. NO. 1 through SEQ. ID. NO. 66; b) fragments of any one of SEQ. ID. NO. 1 through SEQ. ID. NO.
  • fragments have anti-repressor activity; c) sequences that are at least 70% identical in nucleotide sequence to a) or b) wherein said sequences have anti-repressor activity; and d) the complement to any one of a) to c).
  • the invention also provides host cells comprising DNA molecules according to the invention.
  • the invention further provides methods for generating host cells expressing a polypeptide of interest, the method comprising the steps of: introducing into a plurality of precursor host cells a DNA molecule or an expression cassette according to the invention, culturing the cells under conditions selecting for expression of the selectable marker polypeptide, and selecting at least one host cell producing the polypeptide of interest.
  • the invention provides methods for producing a polypeptide of interest, the methods comprising culturing a host cell, said host cell comprising an expression cassette according to the invention, and expressing the polypeptide of interest from the expression cassette.
  • the polypeptide of interest is further isolated from the host cells and/or from the host cell culture medium.
  • FIG. 1 Results with expression constructs according to the invention.
  • the expression construct contains the sequence encoding the polypeptide of interest (exemplified here by d2EGFP) upstream of an IRES, which is upstream of the sequence encoding the selectable marker according to the invention (exemplified here by the zeocin resistance gene, with a TTG startcodon (TTG Zeo) (or in controls with its normal ATG startcodon (ATG Zeo)). See example 1 for details. Dots indicate individual data points; lines indicate the average expression levels; used constructs are indicated on the horizontal axis, and schematically depicted above the graph; vertical axis indicates d2EGFP signal.
  • TTG Zeo TTG startcodon
  • ATG Zeo normal ATG startcodon
  • FIG. 2 Results with tricistronic expression cassettes with dhfr as maintenance marker.
  • the expression construct contains a zeocin selectable marker gene with a TTG startcodon and lacking internal ATG sequences upstream of the sequence encoding the polypeptide of interest (exemplified here by d2EGFP), which is further operably linked via an IRES to a downstream metabolic selection marker dhfr gene (with an ATG startcodon).
  • Dots indicate individual data points (GFP fluorescence signal in Zeo R colonies on vertical axis), lines indicate the average expression levels.
  • the used construct is shown above the graph, conditions are indicated on the horizontal axis (d: day). See example 2 for details.
  • FIG. 3 As Fig 2 , but with dhfr gene having GTG startcodon.
  • FIG. 4 As Fig 2 , but with dhfr gene having TTG startcodon.
  • FIG. 5 Copy numbers in clones with the dhfr enzyme (ATG startcodon), under different conditions. See example 3 for details.
  • FIG. 6 As Fig. 5 , but with dhfr gene having GTG startcodon.
  • FIG. 7 As Fig. 5 , but with dhfr gene having TTG startcodon.
  • the invention provides a DNA molecule according to claim 1.
  • a DNA molecule can be used according to the invention for obtaining eukaryotic host cells expressing high levels of the polypeptide of interest, by selecting for the expression of the selectable marker polypeptide. Subsequently or simultaneously, one or more host cell(s) expressing the polypeptide of interest can be identified, and further used for expression of high levels of the polypeptide of interest.
  • a "monocistronic gene” is defined as a gene capable of providing a RNA molecule that encodes one polypeptide.
  • a "multicistronic transcription unit”, also referred to as multicistronic gene, is defined as a gene capable of providing an RNA molecule that encodes at least two polypeptides.
  • the term “bicistronic gene” is defined as a gene capable of providing a RNA molecule that encodes two polypeptides. A bicistronic gene is therefore encompassed within the definition of a multicistronic gene.
  • a "polypeptide” as used herein comprises at least five amino acids linked by peptide bonds, and can for instance be a protein or a part, such as a subunit, thereof.
  • a “gene” or a “transcription unit” as used in the present invention can comprise chromosomal DNA, cDNA, artificial DNA, combinations thereof, and the like. Transcription units comprising several cistrons are transcribed as a single mRNA.
  • a multicistronic transcription unit according to the invention preferably is a bicistronic transcription unit coding from 5' to 3' for a polypeptide of interest and for a selectable marker polypeptide.
  • the polypeptide of interest is encoded upstream from the coding sequence for the selectable marker polypeptide.
  • the IRES is operably linked to the sequence encoding the selectable marker polypeptide, and hence the selectable marker polypeptide is dependent from the IRES for its translation.
  • the DNA molecules of the invention can be present in the form of double stranded DNA, having with respect to the selectable marker polypeptide and the polypeptide of interest a coding strand and a non-coding strand, the coding strand being the strand with the same sequence as the translated RNA, except for the presence of T instead of U.
  • an AUG startcodon is coded for in the coding strand by an ATG sequence, and the strand containing this ATG sequence corresponding to the AUG startcodon in the RNA is referred to as the coding strand of the DNA.
  • startcodons or translation initiation sequences are in fact present in an RNA molecule, but that these can be considered equally embodied in a DNA molecule coding for such an RNA molecule; hence, wherever the present invention refers to a startcodon or translation initation sequence, the corresponding DNA molecule having the same sequence as the RNA sequence but for the presence of a T instead of a U in the coding strand of said DNA molecule is meant to be included, and vice versa, except where explicitly specified otherwise.
  • a startcodon is for instance an AUG sequence in RNA, but the corresponding ATG sequence in the coding strand of the DNA is referred to as startcodon as well in the present invention.
  • the selectable marker polypeptide and the polypeptide of interest encoded by the multicistronic gene each have their own translation initation sequence, and therefore each have their own startcodon (as well as stopcodon), i.e.,they are encoded by separate open reading frames.
  • selection marker or “selectable marker” is typically used to refer to a gene and/or protein whose presence can be detected directly or indirectly in a cell, for example a polypeptide that inactivates a selection agent and protects the host cell from the agent's lethal or growth-inhibitory effects (e.g. an antibiotic resistance gene and/or protein).
  • selection marker induces fluorescence or a color deposit (e.g. green fluorescent protein (GFP) and derivatives (e.g d2EGFP), luciferase, lacZ, alkaline phosphatase, etc.), which can be used for selecting cells expressing the polypeptide inducing the color deposit, e.g.
  • the selectable marker polypeptide according to the invention provides resistance against lethal and/or growth-inhibitory effects of a selection agent.
  • the selectable marker polypeptide is encoded by the DNA of the invention.
  • the selectable marker polypeptide according to the invention must be functional in a eukaryotic host cell, and hence being capable of being selected for in eukaryotic host cells. Any selectable marker polypeptide fulfilling this criterion can in principle be used according to the present invention.
  • selectable marker polypeptides are well known in the art and routinely used when eukaryotic host cell clones are to be obtained, and several examples are provided herein.
  • a selection marker used for the invention is zeocin.
  • blasticidin is used.
  • selection markers are available and can be used, e.g. neomycin, puromycin, bleomycin, hygromycin, etc.
  • kanamycin is used.
  • the DHFR gene is used as a selectable marker, which can be selected for by methotrexate, especially by increasing the concentration of methotrexate cells can be selected for increased copy numbers of the DHFR gene.
  • the DHFR gene may also be used to complement dhfr-deficiency, e.g.
  • GS glutamine synthetase
  • MSX methionine sulphoximine
  • selectable marker genes that could be used, and their selection agents, are for instance described in table 1 of US patent 5,561,053 , ; see also Kaufman, Methods in Enzymology, 185:537-566 (1990 ), for a review of these.
  • the selectable marker polypeptide is dhfr
  • the host cell in advantageous embodiments is cultured in a culture medium that contains folate and which culture medium is essentially devoid of hypoxanthine and thymidine, and preferably also of glycine.
  • each one preferably contains the coding sequence for a different selectable marker, to allow selection for both multicistronic transcription units.
  • both multicistronic transcription units may be present on a single nucleic acid molecule or alternatively each one may be present on a separate nucleic acid molecule.
  • selection is typically defined as the process of using a selection marker/selectable marker and a selection agent to identify host cells with specific genetic properties (e.g. that the host cell contains a transgene integrated into its genome). It is clear to a person skilled in the art that numerous combinations of selection markers are possible.
  • One antibiotic that is particularly advantageous is zeocin, because the zeocin-resistance protein (zeocin-R) acts by binding the drug and rendering it harmless. Therefore it is easy to titrate the amount of drug that kills cells with low levels of zeocin-R expression, while allowing the high-expressors to survive.
  • antibiotic-resistance proteins in common use are enzymes, and thus act catalytically (not 1:1 with the drug).
  • the antibiotic zeocin is a preferred selection marker.
  • Another preferred selection marker is a 5,6,7,8-tetrahydrofolate synthesizing enzyme (dhfr).
  • dhfr 5,6,7,8-tetrahydrofolate synthesizing enzyme
  • the invention also works with other selection markers.
  • a selectable marker polypeptide according to the invention is the protein that is encoded by the nucleic acid of the invention, which polypeptide can be functionally used for selection, for instance because it provides resistance to a selection agent such as an antibiotic.
  • the DNA encodes a polypeptide that centers resistance to the selection agent, which polypeptide is the selectable marker polypeptide.
  • DNA sequences coding for such selectable marker polypeptides are known, and several examples of wild-type sequences of DNA encoding selectable marker proteins are provided herein (e.g. Figs. 26-32 of WO 2006/048459 ). It will be clear that mutants or derivatives of selectable markers can also be suitably used according to the invention, and are therefore included within the scope of the term 'selectable marker polypeptide', as long as the selectable marker protein is still functional.
  • the gene and protein encoding the resistance to a selection agent is referred to as the 'selectable agent (resistance) gene' or 'selection agent (resistance) protein', respectively, although the official names may be different, e.g. the gene coding for the protein conferring restance to neomycin (as well as to G418 and kanamycin) is often referred to as neomycin (resistance) (or neo r ) gene, while the official name is aminoglycoside 3'-phosphotransferase gene.
  • selectable marker polypeptide it is beneficial to have low levels of expression of the selectable marker polypeptide, so that stringent selection is possible. In the present invention this is brought about by using a selectable marker coding sequence with a non-ATG startcodon. Upon selection, only cells that have nevertheless sufficient levels of selectable marker polypeptide will be selected, meaning that such cells must have sufficient transcription of the multicistronic transcription unit and sufficient translation of the selectable marker polypeptide, which provides a selection for cells where the multicistronic transcription unit has been integrated or otherwise present in the host cells at a place where expression levels from this transcription unit are high.
  • the DNA molecules according to the invention have the coding sequence for the selectable marker polypeptide downstream of the coding sequence for the polypeptide of interest.
  • the multicistronic transcription unit comprises in the 5' to 3' direction (both in the transcribed strand of the DNA and in the resulting transcribed RNA) the sequence encoding the polypeptide of interest and the coding sequence for the selectable marker polypeptide.
  • the IRES is upstream of the coding sequence for the selectable marker polypeptide.
  • the coding region of the gene of interest is preferably translated from the cap-dependent ORF, and the polypeptide of interest is produced in abundance.
  • the selectable marker polypeptide is translated from an IRES.
  • the nucleic acid sequence coding for the selectable marker polypeptide comprises a mutation in the startcodon that decreases the translation initiation efficiency of the selectable marker polypeptide in a eukaryotic host cell.
  • a GTG startcodon or more prefereably a TTG startcodon is engineered into the selectable marker polypeptide.
  • the translation efficiency is lower than that of the corresponding wild-type sequence in the same cell, i.e. the mutation results in less polypeptide per cell per time unit, and hence less selectable marker polypeptide.
  • a translation start sequence is often referred to in the field as 'Kozak sequence', and an optimal Kozak sequence is RCC ATG G, the startcodon underlined, R being a purine, i.e. A or G (see Kozak M, 1986, 1987, 1989, 1990, 1997, 2002).
  • an optimal translation startsequence comprises an optimal startcodon (i.e. ATG) in an optimal context (i.e. the ATG directly preceded by RCC and directly followed by G).
  • the ATG startcodon of the selectable marker polypeptide in the invention is mutated into another codon, which has been reported to provide some translation initiation, for instance to GTG, TTG, CTG, ATT, or ACG (collectively referred to herein as 'non-ATG start codons').
  • the ATG startcodon is mutated into a GTG startcodon. This provides still lower expression levels (lower translation) than with the ATG startcodon intact but in a non-optimal context.
  • the ATG startcodon is mutated to a TTG startcodon, which provides even lower expression levels of the selectable marker polypeptide than with the GTG startcodon (Kozak M, 1986, 1987, 1989, 1990, 1997, 2002; see also examples 9-13 in WO 2006/048459 ).
  • the use of non-ATG startcodons in the coding sequence for a selectable marker polypeptide in a multicistronic transcription unit according to the present invention was not disclosed nor suggested in the prior art and, preferably in combination with chromatin control elements, leads to very high levels of expression of the polypeptide of interest, as also shown in WO 2006/048459 .
  • non-ATG startcodons are preferably directly preceded by nucleotides RCC in positions -3 to -1 and directly followed by a G nucleotide (position +4).
  • RCC nucleotides
  • G nucleotide
  • ATG sequences within the coding sequence for a polypeptide, but excluding the ATG startcodon, are referred to as ⁇ internal ATGs', and if these are in frame with the ORF and therefore code for methionine, the resulting methionine in the polypeptide is referred to as an 'internal methionine'.
  • the coding region (following the startcodon, not necessarily including the startcodon) coding for the selectable marker polypeptide is devoid of any ATG sequence in the coding strand of the DNA, up to (but not including) the startcodon of the polypeptide of interest.
  • WO 2006/048459 discloses how to bring this about and how to test the resulting selectable marker polypeptides for functionality.
  • the selectable marker polypeptide coding sequence is downstream of an IRES and downstream of the coding sequence for the polypeptide of interest, internal ATGs in the sequence encoding the selectable marker polypeptide can remain intact.
  • the translation start sequence of the polypeptide of interest comprises an optimal translation start sequence, i.e. having the consensus sequence RCC ATG G (startcodon underlined). This will result in a very efficient translation of the polypeptide of interest.
  • the stringency of selection can be increased. Fine-tuning of the selection system is thus possible using the multicistronic transcription units according to the invention: for instance using a GTG startcodon for the selection marker polypeptide, only few ribosomes will translate from this startcodon, resulting in low levels of selectable marker protein, and hence a high stringency of selection; using a TTG startcodon even further increases the stringency of selection because even less ribosomes will translate the selectable marker polypeptide from this startcodon.
  • the multicistronic expression units disclosed therein can be used in a very robust selection system, leading to a very large percentage of clones that express the polypeptide of interest at high levels, as desired.
  • the expression levels obtained for the polypeptide of interest appear to be significantly higher than those obtained when an even larger number of colonies are screened using selection systems hitherto known.
  • the selectable marker polypeptide in addition to a decreased translation initiation efficiency, it could be beneficial to also provide for decreased translation elongation efficiency of the selectable marker polypeptide, e.g. by mutating the coding sequence thereof so that it comprises several non-preferred codons of the host cell, in order to further decrease the translation levels of the marker polypeptide and allow still more stringent selection conditions, if desired.
  • the selectable marker polypeptide further comprises a mutation that reduces the activity of the selectable marker polypeptide compared to its wild-type counterpart. This may be used to increase the stringency of selection even further.
  • proline at position 9 in the zeocin resistance polypeptide may be mutated, e.g. to Thr or Phe (see e.g. example 14 of WO 2006/048459 ), and for the neomycin resistance polypeptide, amino acid residue 182 or 261 or both may further be mutated (see e.g. WO 01/32901 ).
  • a so-called spacer sequence is placed downstream of the sequence encoding the startcodon of the selectable marker polypeptide, which spacer sequence preferably is a sequence in frame with the startcodon and encoding a few amino acids, and that does not contain a secondary structure (Kozak, 1990).
  • Such a spacer sequence can be used to further decrease the translation initiation frequency if a secondary structure is present in the RNA (Kozak, 1990) of the selectable marker polypeptide (e.g. for zeocin, possibly for blasticidin), and hence increase the stringency of the selection system according to the invention (see e.g. example 14 of WO 2006/048459 ).
  • any DNA molecules as described but having mutations in the sequence downstream of the first ATG (startcodon) coding for the selectable marker protein can also be used and are thus also encompassed in the invention, as long as the respective encoded selectable marker protein still has activity.
  • startcodon startcodon
  • any silent mutations that do not alter the encoded protein because of the redundancy of the genetic code are also encompassed.
  • Further mutations that lead to conservative amino acid mutations or to other mutations are also encompassed, as long as the encoded protein still has activity, which may or may not be lower than that of the wild-type protein as encoded by the indicated sequences.
  • the encoded protein is at least 70%, preferably at least 80%, more preferably at least 90%, still more preferably at least 95% identical to the proteins encoded by the respective indicated sequences (e.g. as provided in SEQ ID NOs. 68-80 of the sequence listing of the present application). Testing for activity of the selectable marker proteins can be done by routine methods.
  • an expression cassette comprising the DNA molecule according to the invention, having the multicistronic transcription unit.
  • an expression cassette is useful to express sequences of interest, for instance in host cells.
  • An 'expression cassette' as used herein is a nucleic acid sequence comprising at least a promoter functionally linked to a sequence of which expression is desired.
  • an expression cassette further contains transcription termination and polyadenylation sequences. Other regulatory sequences such as enhancers may also be included.
  • the invention provides an expression cassette comprising in the following order: 5'- promoter - multicistronic transcription unit according to the invention, coding for a polypeptide of interest and downstream thereof a selectable marker polypeptide - transcription termination sequence - 3'.
  • the promoter must be capable of functioning in a eukaryotic host cell, i.e. it must be capable of driving transcription of the multicistronic transcription unit.
  • the promoter is thus operably linked to the multicistronic transcription unit.
  • the expression cassette may optionally further contain other elements known in the art, e.g. splice sites to comprise introns, and the like.
  • an intron is present behind the promoter and before the sequence encoding the polypeptide of interest.
  • An IRES is operably linked to the cistron that contains the selectable marker polypeptide coding sequence.
  • a sequence coding for a second selectable marker is present in the multicistronic transcription unit (i.e.
  • said sequence encoding a second selectable marker polypeptide a) has a translation initiation sequence separate from that of the polypeptide of interest, b) is positioned upstream of said sequence encoding a polypeptide of interest, c) has no ATG sequence in the coding strand following the startcodon of said second selectable marker polypeptide up to the startcodon of the polypeptide of interest, and d) has a non-optimal translation start sequence, e.g. a GTG startcodon or a TTG startcodon.
  • a preferred selectable marker polypeptide is a 5,6,7,8-tetrahydrofolate synthesizing enzyme (dhfr). This allows for continuous selection of high levels of expression of the polypeptide of interest, as exemplified in example 2.
  • nucleic acid sequences encoding protein it is well known to those skilled in the art that sequences capable of driving such expression, can be functionally linked to the nucleic acid sequences encoding the protein, resulting in recombinant nucleic acid molecules encoding a protein in expressible format.
  • the expression cassette comprises a multicistronic transcription unit.
  • the promoter sequence is placed upstream of the sequences that should be expressed.
  • Much used expression vectors are available in the art, e.g.
  • Sequences driving expression may include promoters, enhancers and the like, and combinations thereof. These should be capable of functioning in the host cell, thereby driving expression of the nucleic acid sequences that are functionally linked to them.
  • promoters can be used to obtain expression of a gene in host cells. Promoters can be constitutive or regulated, and can be obtained from various sources, including viruses, prokaryotic, or eukaryotic sources, or artificially designed.
  • Expression of nucleic acids of interest may be from the natural promoter or derivative thereof or from an entirely heterologous promoter (Kaufman, 2000). According to the present invention, strong promoters that give high transcription levels in the eukaryotic cells of choice are preferred. Suitable promoters are well known and available to the skilled person, and several are described in WO 2006/048459 (e.g. page 28-29), including the CMV immediate early (IE) promoter (referred to herein as the CMV promoter) (obtainable for instance from pcDNA, Invitrogen), and many others.
  • IE CMV immediate early
  • a DNA molecule according to the invention is part of a vector, e.g. a plasmid.
  • a vector e.g. a plasmid.
  • Such vectors can easily be manipulated by methods well known to the person skilled in the art, and can for instance be designed for being capable of replication in prokaryotic and/or eukaryotic cells.
  • many vectors can directly or in the form of isolated desired fragment therefrom be used for transformation of eukaryotic cells and will integrate in whole or in part into the genome of such cells, resulting in stable host cells comprising the desired nucleic acid in their genome.
  • plasmid or the viral genome is introduced into (eukaryotic host) cells and preferably integrated into their genomes by methods known in the art, and several aspects hereof have been described in WO 2006/048459 (e.g. pag. 30-31),
  • an expression cassette according to the invention further comprises at least one chromatin control element.
  • a 'chromatin control element' as used herein is a collective term for DNA sequences that may somehow have an effect on the chromatin structure and therewith on the expression level and/or stability of expression of transgenes in their vicinity (they function 'in cis', and hence are placed preferably within 5 kb, more preferably within 2 kb, still more preferably within 1 kb from the transgene) within eukaryotic cells.
  • Such elements have sometimes been used to increase the number of clones having desired levels of transgene expression.
  • WO 2006/048459 e.g.
  • chromatin control elements are chosen from the group consisting of matrix or scaffold attachment regions (MARs/SARs), insulators such as the beta-globin insulator element (5' HS4 of the chicken beta-globin locus), scs, scs', and the like, a ubiquitous chromatin opening element (UCOE), and anti-repressor sequences (also referred to as 'STAR' sequences).
  • MARs/SARs matrix or scaffold attachment regions
  • insulators such as the beta-globin insulator element (5' HS4 of the chicken beta-globin locus), scs, scs', and the like, a ubiquitous chromatin opening element (UCOE), and anti-repressor sequences (also referred to as 'STAR' sequences).
  • said chromatin control element is an anti-repressor sequence, preferably chosen from the group consisting of: a) any one SEQ. ID. NO. 1 through SEQ. ID. NO. 66; b) fragments of any one of SEQ. ID. NO. 1 through SEQ. ID. NO. 66, wherein said fragments have anti-repressor activity ('functional fragments'); c) sequences that are at least 70% identical in nucleotide sequence to a) or b) wherein said sequences have anti-repressor activity ('functional derivatives'); and d) the complement to any one of a) to c).
  • said chromatin control element is chosen from the group consisting of STAR67 (SEQ. ID. NO.
  • STAR7 SEQ. ID. NO. 7
  • STAR9 SEQ. ID. NO. 9
  • STAR17 SEQ. ID. NO. 17
  • STAR27 SEQ. ID. NO. 27
  • STAR29 SEQ. ID. NO. 29
  • STAR43 SEQ. ID. NO. 43
  • STAR44 SEQ. ID. NO. 44
  • STAR45 SEQ. ID. NO. 45
  • STAR47 SEQ. ID. NO. 47
  • STAR61 SEQ. ID. NO. 61
  • said STAR sequence is STAR 67 (SEQ. ID. NO. 66) or a functional fragment or derivative thereof.
  • STAR 67 or a functional fragment or derivative thereof is positioned upstream of a promoter driving expression of the multicistronic transcription unit.
  • the expression cassettes according to the invention are flanked on both sides by at least one anti-repressor sequence, e.g. by one of SEQ. ID. NO. I through SEQ. ID. NO. 65 on both sides, preferably each with the 3' end of these sequences facing the transcription unit.
  • expression cassettes are provided according to the invention, comprising in 5' to 3' order: anti-repressor sequence A - anti-repressor sequence B - [promoter - multicistronic transcription unit according to the invention (encoding the polypeptide of interest and downstream thereof the functional selectable marker protein) - transcription termination sequence] - anti-repressor sequence C, wherein A, B and C may be the same or different.
  • anti-repressor sequences sequences having anti-repressor activity (anti-repressor sequences) and characteristics thereof, as well as functional fragments or derivatives thereof, and structural and functional definitions thereof, and methods for obtaining and using them, which sequences are useful for the present invention, have been described in WO 2006/048459 (e.g. page 34-38), .
  • both expression cassettes are multicistronic expression cassettes according to the invention, each coding for a different selectable marker protein, so that selection for both expression cassettes is possible.
  • This embodiment has proven to give good results, e.g. for the expression of the heavy and light chain of antibodies.
  • both expression cassettes may be placed on one nucleic acid molecule or both may be present on a separate nucleic acid molecule, before they are introduced into host cells.
  • An advantage of placing them on one nucleic acid molecule is that the two expression cassettes are present in a single predetermined ratio (e.g. 1:1) when introduced into host cells.
  • At least one of the expression cassettes preferably at least one of the expression cassettes, but more preferably each of them, comprises a chromatin control element, more preferably an anti-repressor sequence.
  • the different subunits or parts of a multimeric protein are present on a single expression cassette.
  • transcription units or expression cassettes according to the invention are provided, further comprising a transcription pause (TRAP) sequence, essentially as described on page 40-41 of WO 2006/048459 ,
  • TRAP transcription pause
  • SEQ. ID. NO. 81 One non-limiting example of a TRAP sequence is given in SEQ. ID. NO. 81. Examples of other TRAP sequences, methods to find these, and uses thereof have been described in WO 2004/055215 .
  • DNA molecules comprising multicistronic transcription units and/or expression cassettes according to the present invention can be used for improving expression of nucleic acid, preferably in host cells.
  • the terms "cell”/"host cell” and “cell line”/"host cell line” are respectively typically defined as a cell and homogeneous populations thereof that can be maintained in cell culture by methods known in the art, and that have the ability to express heterologous or Homologous proteins.
  • exemplary host cells that can be used have been described in WO 2006/048459 (e.g. page 41-42), and such cells include for instance mammalian cells, including but not limited to CHO cells, e.g. CHO-K1, CHO-S, CHO-DG44, CHO-DUKXB11, including CHO cells having a dhfr - phenotype, as well as myeloma cells (e.g. Sp2/0, NS0), HEK 293 cells, and PER.C6 cells.
  • mammalian cells including but not limited to CHO cells, e.g. CHO-K1, CHO-S, CHO-DG44, CHO-DUKXB11, including CHO cells having a dhfr - phenotype, as well as myeloma cells (e.g. Sp2/0, NS0), HEK 293 cells, and PER.C6 cells.
  • Such eukaryotic host cells can express desired polypeptides, and are often used for that purpose. They can be obtained by introduction of a DNA molecule of the invention, preferably in the form of an expression cassette, into the cells.
  • the expression cassette is integrated in the genome of the host cells, which can be in different positions in various host cells, and selection will provide for a clone where the transgene is integrated in a suitable position, leading to a host cell clone with desired properties in terms of expression levels, stability, growth characteristics, and the like.
  • the multicistronic transcription unit may be targeted or randomly selected for integration into a chromosomal region that is transcriptionally active, e.g. behind a promoter present in the genome.
  • Selection for cells containing the DNA of the invention can be performed by selecting for the selectable marker polypeptide, using routine methods known by the person skilled in the art.
  • an expression cassette according to the invention can be generated in situ, i.e. within the genome of the host cells.
  • the host cells are from a stable clone that can be selected and propagated according to standard procedures known to the person skilled in the art.
  • a culture of such a clone is capable of producing polypeptide of interest, if the cells comprise the multicistronic transcription unit of the invention.
  • nucleic acid that is to be expressed in a cell can be done by one of several methods, which as such are known to the person skilled in the art, also dependent on the format of the nucleic acid to be introduced. Said methods include but are not limited to transfection, infection, injection, transformation, and the like. Suitable host cells that express the polypeptide of interest can be obtained by selection.
  • the DNA molecule comprising the multicistronic transcription unit of the invention preferably in the form of an expression cassette, is integrated into the genome of the eukaryotic host cell according to the invention. This will provide for stable inheritance of the multicistronic transcription unit.
  • Selection for the presence of the selectable marker polypeptide, and hence for expression, can be performed during the initial obtaining of the cells.
  • selection agent is present in the culture medium at least part of the time during the culturing, either in sufficient concentrations to select for cells expressing the selectable marker polypeptide or in lower concentrations. In preferred embodiments, selection agent is no longer present in the culture medium during the production phase when the polypeptide is expressed.
  • a polypeptide of interest according to the invention can be any protein, and may be a monomeric protein or a (part of a) multimeric protein.
  • a multimeric protein comprises at least two polypeptide chains.
  • Non-limiting examples of a protein of interest according to the invention are enzymes, hormones, immunoglobulin chains, therapeutic proteins like anti-cancer proteins, blood coagulation proteins such as Factor VIII, multi-functional proteins, such as erythropoietin, diagnostic proteins, or proteins or fragments thereof useful for vaccination purposes, all known to the person skilled in the art.
  • an expression cassette of the invention encodes an immunoglobulin heavy or light chain or an antigen binding part, derivative and/or analogue thereof.
  • a protein expression unit according to the invention is provided, wherein said protein of interest is an immunoglobulin heavy chain.
  • a protein expression unit according to the invention is provided, wherein said protein of interest is an immunoglobulin light chain.
  • an immunoglobulin may be encoded by the heavy and light chains on different expression cassettes, or on a single expression cassette.
  • the heavy and light chain are each present on a separate expression cassette, each having its own promoter (which may be the same or different for the two expression cassettes), each comprising a multicistronic transcription unit according to the invention, the heavy and light chain being the polypeptide of interest, and preferably each coding for a different selectable marker protein, so that selection for both heavy and light chain expression cassette can be performed when the expression cassettes are introduced and/or present in a eukaryotic host cell.
  • the polypeptide of interest may be from any source, and in certain embodiments is a mammalian protein, an artificial protein (e.g. a fusion protein or mutated protein), and preferably is a human protein.
  • an artificial protein e.g. a fusion protein or mutated protein
  • the configurations of the expression cassettes of the present invention may also be used when the ultimate goal is not the production of a polypeptide of interest, but the RNA itself, for instance for producing increased quantities of RNA from an expression cassette, which may be used for purposes of regulating other genes (e.g. RNAi, antisense RNA), gene therapy, in vitro protein production, etc.
  • the invention provides a method for generating a host cell expressing a polypeptide of interest, the method comprising introducing into a plurality of precursor cells a DNA molecule or an expression cassette according to the invention, culturing the generated cells under selection conditions and selecting at least one host cell producing the polypeptide of interest.
  • Advantages of this novel method are similar to those described for the alternative method disclosed in WO 2006/048459 (e.g. page 46-47),
  • the selection system of the invention nevertheless can be combined with amplification methods to even further improve expression levels.
  • This can for instance be accomplished by amplification of a co-integrated dhfr gene using methotrexate, for instance by placing dhfr on the same nucleic acid molecule as the multicistronic transcription unit of the invention, or by cotransfection when dhfr is on a separate DNA molecule.
  • the dhfr gene can also be part of a multicistronic expression unit of the invention.
  • the invention also provides methods for producing one or more polypeptides of interest, the method comprising culturing host cells of the invention.
  • Culturing a cell is done to enable it to metabolize, and/or grow and/or divide and/or produce recombinant proteins of interest. This can be accomplished by methods well known to persons skilled in the art, and includes but is not limited to providing nutrients for the cell.
  • the methods comprise growth adhering to surfaces, growth in suspension, or combinations thereof Culturing can be done for instance in dishes, roller bottles or in bioreactors, using batch, fed-batch, continuous systems such as perfusion systems, and the like.
  • the expressed protein is collected (isolated), either from the cells or from the culture medium or from both. It may then be further purified using known methods, e.g. filtration, column chromatography, etc, by methods generally known to the person skilled in the art.
  • the selection method according to the present invention works in the absence of chromatin control elements, but improved results are obtained when the multicistronic expression units are provided with such elements.
  • the selection method according to the present invention works particularly well when an expression cassette according to the invention, comprising at least one anti-repressor sequence is used. Depending on the selection agent and conditions, the selection can in certain cases be made so stringent, that only very few or even no host cells survive the selection, unless anti-repressor sequences are present.
  • the combination of the novel selection method and anti-repressor sequences provides a very attractive method to obtain only limited numbers of colonies with a greatly improved chance of high expression of the polypeptide of interest therein, while at the same time the obtained clones comprising the expression cassettes with anti-repressor sequences provide for stable expression of the polypeptide of interest, i.e. they are less prone to silencing or other mechanisms of lowering expression than conventional expression cassettes.
  • the invention provides a multicistronic transcription unit having an alternative configuration compared to the configuration disclosed in WO 2006/048459 : in the alternative configuration of the present invention, the sequence coding for the polypeptide of interest is upstream of the sequence coding for the selectable marker polypeptide, and the selectable marker polypeptide is operably linked to a cap-independent translation initiation sequence, preferably an internal ribosome entry site (IRES).
  • a cap-independent translation initiation sequence preferably an internal ribosome entry site (IRES).
  • IRS internal ribosome entry site
  • the startcodon of the selectable marker polypeptide is changed into a non-ATG startcodon, to further decrease the translation initiation rate for the selectable marker.
  • This therefore leads to a desired decreased level of expression of the selectable marker polypeptide, and can result in highly effective selection host cells expressing high levels of the polypeptide of interest, as with the embodiments disclosed in WO 2006/048459 .
  • One potential advantage of this alternative aspect of the present invention compared to the embodiments outlined in WO 2006/048459 , is that the coding sequence of the selectable marker polypeptide needs no further modification of internal ATG sequences, because any internal ATG sequences therein can remain intact since they are no longer relevant for translation of further downstream polypeptides.
  • the coding sequence for the selectable marker polypeptide in the DNA molecules of the present invention is under translational control of the IRES, whereas the coding sequence for the protein of interest is preferably translated in a cap-dependent manner.
  • the coding sequence for the polypeptide of interest comprises a stopcodon, so that translation of the first cistron ends upstream of the IRES, which IRES is operably linked to the second cistron.
  • multicistronic expression units can be advantageously varied along the same lines as for the multicistronic expression units having an opposite order of the coding sequences for the polypeptide of interest and the selectable marker polypeptide (i.e. the multicistronic transcription units of WO 2006/048459 ).
  • the preferred startcodons for the selectable marker polypeptide, the incorporation into expression cassettes, the host cells, the promoters, the presence of chromatin control elements, etc. can be varied and used in preferred embodiments as described supra. Also the use of these multicistronic expression units and expression cassettes is as described supra.
  • this aspect is really an alternative to the means and methods described in WO 2006/048459 , with the main difference being that the order of the polypeptides in the multicistronic expression units is reversed, and that an IRES is now required for the translation of the selectable marker polypeptide.
  • an "internal ribosome entry site” or “IRES” refers to an element that promotes direct internal ribosome entry to the initiation codon, such as normally an ATG, but in this invention preferably GTG or TTG, of a cistron (a protein encoding region), thereby leading to the cap-independent translation of the gene.
  • initiation codon such as normally an ATG, but in this invention preferably GTG or TTG, of a cistron (a protein encoding region), thereby leading to the cap-independent translation of the gene.
  • the present invention encompasses the use of any cap-independent translation initiation sequence, in particular any IRES element that is able to promote direct internal ribosome entry to the initiation codon of a cistron.
  • Under translational control of an IRES as used herein means that translation is associated with the IRES and proceeds in a cap-independent manner.
  • the term “IRES” encompasses functional variations of IRES sequences as long as the variation is able to promote direct internal ribosome entry to the initiation codon of a cistron.
  • cistron refers to a polynucleotide sequence, or gene, of a protein, polypeptide, or peptide of interest.
  • “Operably linked” refers to a situation where the components described are in a relationship permitting them to function in their intended manner.
  • a promoter "operably linked" to a cistron is ligated in such a manner that expression of the cistron is achieved under conditions compatible with the promoter.
  • a nucleotide sequence of an IRES operably linked to a cistron is ligated in such a manner that translation of the cistron is achieved under conditions compatible with the IRES.
  • IRES Internal ribosome binding site
  • IRES permits two or more proteins to be produced from a single RNA molecule (the first protein is translated by ribosomes that bind the RNA at the cap structure of its 5' terminus, (Martinez-Salas, 1999)).
  • Translation of proteins from IRES elements is less efficient than cap-dependent translation: the amount of protein from IRES-dependent open reading frames (ORFs) ranges from less than 20% to 50% of the amount from the first ORF (Mizuguchi et al., 2000).
  • ORFs open reading frames
  • IRES elements can attenuate their activity, and lower the expression from the IRES-dependent ORFs to below 10% of the first ORF (Lopez de Quinto & Martinez-Salas, 1998, Rees et al., 1996). It is therefore clear to a person skilled in the art that changes to the IRES can be made without altering the essence of the function of the IRES (hence, providing a protein translation initiation site with a reduced translation efficiency), resulting in a modified IRES. Use of a modified IRES which is still capable of providing a small percentage of translation (compared to a 5' cap translation) is therefore also included in this invention.
  • the present invention uses non-ATG startcodons to significantly further reduce translation initation of the selectable marker ORF, therewith further improving the chances of obtaining a preferred host cell, i.e. a host cell expressing high levels of recombinant protein of interest.
  • US patents 5,648,267 and 5,733,779 describe the use of a dominant selectable marker sequence with an impaired consensus Kozak sequence ([Py]xxATG[Py], wherein [Py] is a pyrimidine nucleotide (i.e. C or T), x is a nucleotide (i.e. G, A, T, or C), and the ATG startcodon is underlined).
  • US patent 6,107,477 describes the use of a non-optimal Kozak sequence (AGATCTTT ATG GACC, wherein the ATG startcodon is underlined) for a selectable marker gene. None of these patents describes the use of a non-ATG startcodon, nor provides any suggestion to do so.
  • the invention also provides a DNA molecule comprising a sequence coding for a selectable marker polypeptide operably linked to an IRES sequence, wherein the coding sequence coding for the selectable marker polypeptide comprises a translation start sequence selected from the group consisting of: a) a GTG start codon; b) a TTG start codon; c) a CTG start codon; d) a ATT start codon; and e) a ACG start codon.
  • the mammalian 5,6,7,8 tetrahydrofolate synthesizing enzyme dihydrofolate reductase can be used as a selection marker in cells that have a dhfr phenotype (e.g. CHO-DG44 cells), by omitting hypoxanthine and thymidine (and preferably also glycine) from the culture medium and including folate (or (dihydro)folic acid) into the culture medium (Simonsen et al, 1988).
  • a dhfr phenotype e.g. CHO-DG44 cells
  • the dhfr gene can for instance be derived from the mouse genome or mouse cDNA and can be used according to the invention, preferably by providing it with a GTG or TTG startcodon (see SEQ. ID. NO. 73 for the sequence of the dhfr gene).
  • a GTG or TTG startcodon see SEQ. ID. NO. 73 for the sequence of the dhfr gene.
  • the indicated component is present at a concentration of less than 0.1 % of the concentration of that component that is normally used in the culture medium for a certain cell type.
  • the indicated component is absent from the culture medium.
  • a culture medium lacking the indicated component can be prepared according to standard methods by the skilled person or can be obtained from commercial media suppliers.
  • a potential advantage of the use of these types of metabolic enzymes as selectable marker polypeptides is that they can be used to keep the multicistronic transcription units under continuous selection, which may result in higher expression of the polypeptide of interest.
  • the invention uses the dhfr metabolic selection marker as an additional selection marker in a multicistronic transcription unit according to the invention.
  • selection of host cell clones with high expression is first established by use of for instance an antibiotic selection marker, e.g. zeocin, neomycin, etc, the coding sequences of which will have a GTG or TTG startcodon according to the invention.
  • an antibiotic selection marker e.g. zeocin, neomycin, etc
  • the antibiotic selection is discontinued, and now continuous or intermittent selection using the metabolic enzyme selection marker can be performed by culturing the cells in the medium lacking the appropriate identified components described supra and containing the appropriate precursor components described supra.
  • the metabolic selection marker is operably linked to an IRES, and can have its normal ATG content, and the startcodon can be suitably chosen from GTG or TTG.
  • the multicistronic transcription units in this aspect are at least tricistronic.
  • Example 1 describes the selection system with the multicistronic transcription unit of the present invention, and it will be clear that the variations described in examples 8-26 of WO 2006/048459 , can also be applied and tested for the multicistronic transcription units of the present application. The same holds for those of examples 20-27 of US 2006/0195935 .
  • Example 1 Stringent selection by placing a modified Zeocin resistance gene behind an IRES sequence
  • Examples 8-26 of WO 2006/048459 have shown a selection system where a sequence encoding a selectable marker protein is upstream of a sequence encoding a protein of interest in a multicistonic transcription unit, and wherein the translation initiation sequence of the selectable marker is non-optimal, and wherein further internal ATGs have been removed from the selectable marker coding sequence.
  • This system results in a high stringency selection system. For instance the Zeo selection marker wherein the translation initiation codon is changed into TTG was shown to give very high selection stringency, and very high levels of expression of the protein of interest encoded downstream.
  • the selection marker e.g. Zeo
  • the selection marker is placed downstream from an IRES sequence.
  • the Zeo startcodon is the optimal ATG.
  • IRES-TTG Zeo we tested whether changing the Zeo ATG startcodon into for instance TTG (referred to as IRES-TTG Zeo) resuls in increased selection stringencies compared to the usual IRES-ATG Zeo.
  • the used constructs are schematically shown in FIG. 1 .
  • the control construct consisted of a CMV promoter, the d2EGFP gene, an IRES sequence (the sequence of the used IRES (Rees et al, 1996) in this example was:
  • the marker can be varied along the same lines of examples 8-26 of WO 2006/048459 and 20-27 of US 2006/0195935 .
  • a GTG startcodon can be used, and the marker can be changed from Zeo into a different, marker, e.g. Neo, Blas, dhfr, puro, etc, all with either GTG or TTG as startcodon.
  • the STAR elements can be varied by using different STAR sequences or different placement thereof, or by substituting them for other chromatin control elements, e.g. MAR sequences. This leads to improvements over the prior art selection systems having an IRES with a marker with a normal ATG startcodon.
  • a modified Neomycin resistance gene is placed downstream of an IRES sequence.
  • the modification consists of a replacement of the ATG translation initiation codon of the Neo coding sequence by a TTG translation initiation codon, creating TTG Neo.
  • the CMV-d2EGF-IRES-TTG Neo construct either surrounded by STAR elements or not, is transfected to CHO-K1 cells. Colonies are picked, cells are propagated and d2EGFP values are measured.
  • This leads to improvement over the known selection system having Neo with an ATG startcodon downstream of an IRES ('IRES-ATG Neo'). The improvement is especially apparent when the TTG Neo construct comprises STAR elements.
  • Example 2 Stability of expression by placing a modified dhfr gene behind an IRES sequence
  • TTG selection marker upstream of the reporter gene and coupled a GTG or TTG modified metabolic marker with an IRES to the reporter gene.
  • Different selection marker genes can be used, such as the Zeocin and neomycin resistance genes, as well as the dhfr gene.
  • TTG Zeo modified Zeocin resistance gene
  • IRES IRES
  • Active selection pressure appears beneficial to keep the protein expression levels in a TTG Zeo selected colony at the same high level over a prolonged period of time. This can for instance be accomplished by keeping a minimal amount of Zeocin in the culture medium, but this is not favoured in industrial settings for economic and potentially for regulatory purposes (Zeocin is both toxic and expensive).
  • Another approach is to couple the gene of interest to a selection marker that is an enzyme that metabolizes one or more essential steps in a metabolic pathway.
  • a selection marker that is an enzyme that metabolizes one or more essential steps in a metabolic pathway.
  • essential is meant that the cell is not able to synthesize specific essential metabolic building blocks itself, implying that these building blocks have to be present in the culture medium in order to allow the cell to survive.
  • Well-known examples are the essential amino acids that cannot be synthesized by a mammalian cell and that need to be present in the culture medium to allow the cell to survive.
  • Another example is related to the 5,6,7,8-tetrahydrofolate synthesizing dhfr gene. The corresponding dhfr protein is an enzyme in the folate pathway.
  • the dhfr protein specifically converts folate into 5,6,7,8-tetrahydrofolate, a methyl group shuttle required for the de novo synthesis of purines (Hypoxanthine), thymidylic acid (Thymidine), and the amino acid Glycine.
  • the non-toxic substance folate has to be present in the culture medium (Urlaub et al, 1980).
  • the medium has to lack hypoxanthine and thymidine, since when these are available for the cell, the need for the dhfr enzyme is bypassed.
  • CHO-DG44 cells lack the dhfr gene and these cells therefore need glycine, hypoxanthine and thymidine in the culture medium to survive.
  • the cell can convert folate into 5,6,7,8-tetrahydrofolate, and can thus survive in this culture medium.
  • This principle has been used for many years as selection methodology to create stably transfected mammalian cell lines.
  • TTG Zeo selection marker was placed upstream of the d2EGFP reporter gene and the dhfr selection marker downstream of the d2EGFP gene, coupled through an IRES sequence ( Fig. 2 ). These constructs were flanked with STARs 7/67/7. Three versions of these constructs were made: ATG dhfr, GTG dhfr or TTG dhfr, each name indicating the startcodon used for the dhfr gene. The constructs were transfected to CHO-DG44 cells.
  • DNA was transfected using Lipofectamine 2000 (Invitrogen) and cells were grown in the presence of 400 ⁇ g/ml Zeocin in IMDM medium (Gibco) + 10% FBS (Gibco) + HT-supplement.
  • the average d2EGFP value in 14 TTG Zeo IRES ATG dhfr clones was 341 (day 1), when measured in the presence of 400 ⁇ g/ml Zeocin ( Fig. 2 ). After these measurements the cells were split and further cultured under three conditions:
  • condition 1 the cells are under Zeocin selection pressure only, in condition 2 the cells are NOT under any selection pressure and in condition 3 the cells remain under DHFR selection pressure.
  • condition 3 requires continuous expression of the dhfr gene to allow expression of the dhfr protein and cell survival as a result.
  • TTG Zeo IRES GTG dhfr construct The average d2EGFP value in 15 TTG Zeo IRES GTG dhfr clones was 455 (day 1), when measured in the presence of 400 ⁇ g/ml Zeocin ( Fig. 3 ). After these measurements the cells were split and further cultured under the above described three conditions. After 65 days we again measured the d2EGFP values. The average d2EGFP value in the TTG Zeo IRES GTG dhfr clones under Zeocin selection was now 356 ( Fig. 3 ).
  • the average d2EGFP value in the TTG Zeo IRES GTG dhfr clones without Zeocin selection and with HT supplement was 39 ( Fig. 3 ).
  • the average d2EGFP value in the TTG Zeo IRES GTG dhfr clones without Zeocin selection and without HT supplement was 705 ( Fig. 3 ).
  • TTG Zeo IRES TTG dhfr construct The average d2EGFP value in 18 TTG Zeo IRES TTG dhfr clones was 531 (day 1), when measured in the presence of 400 ⁇ g/ml Zeocin ( Fig. 4 ). After these measurements the cells were split and further cultured under the above described three conditions. After 65 days we again measured the d2EGFP values. The average d2EGFP value in the TTG Zeo IRES TTG dhfr clones under Zeocin selection was now 324 ( Fig. 4 ).
  • the average d2EGFP value in the TTG Zeo IRES TTG dhfr clones without Zeocin selection and in the presence of HT supplement was 33 ( Fig. 4 ).
  • the average d2EGFP value in the TTG Zeo IRES TTG dhfr clones without Zeocin selection and without HT supplement was 1124 ( Fig. 4 ).
  • Example 3 Increased expression by placing a modified dhfr gene behind a weakened IRES sequence is not the result of gene amplification
  • dhfr gene as a selection marker in the prior art often relied on amplification of the dhfr gene.
  • a toxic agent, methotrexate was used in such systems to amplify the dhfr gene, and concomitantly therewith the desired transgene, of which up to many thousands of copies could be found integrated into the genome of CHO cells after such amplification.
  • methotrexate was used in such systems to amplify the dhfr gene, and concomitantly therewith the desired transgene, of which up to many thousands of copies could be found integrated into the genome of CHO cells after such amplification.
  • these high copy numbers lead to high expression levels, they are also considered a disadvantage because so many copies can lead to increased genomic instability, and further removal of methotrexate from the culture medium leads to rapid removal of many of the amplified loci.
  • the average d2EGFP copy number in the TTG Zeo IRES ATG dhfr clones under Zeocin selection was 86 (condition 1)( Fig. 5 ).
  • the average d2EGFP copy number in the TTG Zeo IRES ATG dhfr clones without Zeocin selection and in the presence of HT supplement was 53 (condition 2)( Fig. 5 ).
  • the average d2EGFP copy number in the TTG Zeo IRES ATG dhfr clones without Zeocin selection and without HT supplement was 59 (condition 3)( Fig. 5 ).
  • the average d2EGFP copy number in the TTG Zeo IRES GTG dhfr clones under Zeocin selection was 23 (condition 1)( Fig. 6 ).
  • the average d2EGFP copy number in the TTG Zeo IRES GTG dhfr clones without Zeocin selection and in the presence of HT supplement was 14 (condition 2)( Fig. 6 ).
  • the average d2EGFP copy number in the TTG Zeo IRES GTG dhfr clones without Zeocin selection and without HT supplement was 37 (condition 3)( Fig. 6 ).
  • the average d2EGFP copy number in the TTG Zeo IRES TTG dhfr clones under Zeocin selection was 33 (condition 1)( Fig. 7 ).
  • the average d2EGFP copy number in the TTG Zeo IRES TTG dhfr clones without Zeocin selection and in the presence of HT supplement was 26 (condition 2)( Fig. 7 ).
  • the average d2EGFP copy number in the TTG Zeo IRES TTG dhfr clones without Zeocin selection and without.HT supplement was 32 (condition 3)( Fig. 7 ).

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EP07712283A EP1987150B1 (en) 2006-02-21 2007-02-21 Selection of host cells expressing protein at high levels
PL07712283T PL1987150T3 (pl) 2006-02-21 2007-02-21 Selekcja komórek gospodarza wyrażających białka na wysokim poziomach
SI200730698T SI1987150T1 (sl) 2006-02-21 2007-02-21 Selekcija gostiteljskih celic, ki eksprimirajo protein pri visokih nivojih

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US20060195935A1 (en) 2004-11-08 2006-08-31 Chromagenics B.V. Selection of host cells expressing protein at high levels
US8039230B2 (en) 2004-11-08 2011-10-18 Chromagenics B.V. Selection of host cells expressing protein at high levels
US8999667B2 (en) 2004-11-08 2015-04-07 Chromagenics B.V. Selection of host cells expressing protein at high levels
US7968700B2 (en) 2006-03-20 2011-06-28 Chromagenics B.V. Expression augmenting DNA fragments, use thereof, and methods for finding thereof
KR101655492B1 (ko) 2008-12-22 2016-09-07 국립대학법인 홋가이도 다이가쿠 동물세포를 사용해서 외래유전자 유래 단백질을 대량으로 생산하기 위한 발현 벡터, 및 그의 이용
MX2011009025A (es) 2009-02-27 2011-09-28 Novartis Ag Sistema de vector de expresion que comprende dos marcadores de seleccion.
US8765370B2 (en) * 2009-06-11 2014-07-01 Scinopharm Taiwan, Ltd Inhibition-based high-throughput screen strategy for cell clones
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WO2011113841A1 (en) * 2010-03-16 2011-09-22 Robert Steinfeld Eukaryotic vector
CN103037840A (zh) * 2010-04-21 2013-04-10 国立大学法人北海道大学 具有核内转运性的脂质膜结构体
CN103038352B (zh) * 2010-06-15 2015-12-16 萨拉基尼克有限公司 用于增强基因表达的新型基因间元件
WO2012030218A1 (en) 2010-09-01 2012-03-08 Cellagenics B.V. Nucleic acid fragments from a ribosomal protein promoter for enhancing gene expression
DK3412684T3 (da) 2013-07-31 2022-07-04 Novartis Ag Nye selektionsvektorer og fremgangsmåder til selektering af eukaryotiske værtsceller
CN104531699B (zh) * 2014-10-09 2017-03-15 河南农业大学 一种增强外源基因表达的猪的ucoe调控元件片段
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JP2021532439A (ja) * 2018-07-30 2021-11-25 ナンジン、ジェンスクリプト、バイオテック、カンパニー、リミテッドNanjing Genscript Biotech Co., Ltd. コドン最適化
US20210292752A1 (en) * 2018-08-13 2021-09-23 Spiber Inc. Method for Isolating or Identifying Cell, and Cell Mass
CN110484563B (zh) * 2019-07-25 2023-04-07 新乡医学院 哺乳动物细胞组合表达载体、表达系统、制备方法和应用
KR20210080071A (ko) * 2019-12-20 2021-06-30 (주)셀트리온 목적 단백질의 고발현을 위한 인트론을 포함하는 발현 카세트 및 이의 이용

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